Use of the Antifungal Ilicicolin H in Agriculture

ABSTRACT

The present invention is concerned with the use of the natural product Ilicicolin H as an agricultural fungicide, compositions comprising Ilicicolin H, and the use of such compositions to treat, control or prevent fungal infection in agricultural products.

PRIORITY

This application claims the benefit of U.S. Provisional Application No.62/248,371, filed Oct. 30, 2015, the disclosure of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is concerned with the use of the natural productIlicicolin H as an agricultural fungicide. The invention is alsoconcerned with compositions comprising Ilicicolin H, their preparation,and methods of using such compositions to control agricultural pestsincluding, but not limited to, fungi selected from the group consistingof Botryotinia fuckeliana, Glomerella lagenarium, Mycosphaerellaarachidis, Zymoseptoria tritici, Gaeumannomyces graminis, Monographellanivalis, Thanatephorus cucumeris, Sclerotinia sclerotiorum, and Pucciniarecondita.

BACKGROUND OF THE INVENTION

In the agricultural industry there is a need to control plant pathogensthat would otherwise destroy crops and reduce yields. Farmers havetraditionally employed a variety of methodologies to control thesepests, one of which has been the use of compounds with antimicrobialactivity, called fungicides, to protect the plants and prevent cropdamage. There are many types of plant diseases, and foreignmicroorganisms can be introduced into new areas causing diseaseoutbreaks. Current fungicides have traditionally been chemicallysynthesized compounds that have limited spectrum of activity and oftenrequire repeated usage creating a cause for environmental concern. Theseconcerns include contamination of foodstuffs, soil, surface and groundwater and their impact on native microbial and insect populations. Inaddition, pathogens have been able to develop resistance to conventionalfungicides, and as a result the industry is consistently searching fornew chemical compounds with new modes of action to combat diseaseresistance.

Ilicicolin H is one of several natural secondary metabolites made byimperfect fungi. These metabolites generally act as protectants for thefungus. Ilicicolin H is produced by strains of Cylindrocladium iliciola(Hayakawa et al., “Ilicicolins, antibiotics from Cylindrocladiumilicicola”, Journal of Antibiotics 1971, 24, 653-4) and Fusarium sp.(Shao et al., “Metabolites of endophytic fungus Fusarium from Spartinaalterniflora”, Zhongguo Tianran Yaowu 2007, 5, 108-111), and Gliocladiumroseum (Junker et al., “Scale-Up Studies on a Defined Medium Process forPilot Plant Production of Ilicicolin by Gliocladium roseum,”Biotechnology Progress 2001, 17, 278-286).

Ilicicolin H is an antibiotic that was first identified to have activityagainst the human pathogen Candida albicans and was investigated for usein the treatment of complications to HIV infection in humans(Guetierrez-Cirlos et al., “Inhibition of the Yeast Cytochrome bc1Complex by Ilicicolin H, a Novel Inhibitor That Acts at the Qn Site ofthe bc1Complex,” Journal of Biological Chemistry 2004, 279, 8708-8714).Ilicicolin H whose structure is shown below is a5-(4-hydroxyphenyl)-2-pyridone with a bicyclic decalin system. Thecompound inhibits the mitochondrial respiration of certain fungi byinhibiting the cytochrome bc1 complex. Ilicicolin H has low cyctotoxity(HeLa cells, ED₅₀=2 ug/ml) and low acute toxicity in mice (Hayakawa etal., “Ilicicolins, antibiotics from Cylindrocladium ilicicola,” Journalof Antibiotics 1971, 24, 653-4). However, Ilicicolin H has notpreviously been shown to be active against plant pathogenic fungi.

It has now been found that Ilicicolin H is useful as an agriculturalfungicide to treat, prevent, or control fungal infections inagricultural products, such as plants and seeds.

One aspect of the present invention is concerned with agriculturalcompositions of Ilicicolin H comprising an effective amount of theantifungal compound and an agriculturally acceptable carrier. Suchcompositions may additionally comprise one or more excipients selectedfrom the group consisting of (a) one or more diluents, (b) one or morebinders or binding agents, (c) one or more dispersing agents, (d) one ormore emulsifying agents, (e) one or more surfactants or wetting agents,(f) one or more sticking agents, (g) one or more thickening agents, and(h) a pH adjuster. In one embodiment the surfactant is an anionicsurfactant. In a second embodiment the surfactant is a non-ionicsurfactant.

Ilicicolin H and agricultural compositions of Ilicicolin H of thepresent invention may also be used in combination with one or more otheragents useful to treat, prevent, or control agricultural pests in thefield. In such combinations the compositions of the present inventionand other active agents may be administered separately or inconjunction. In addition, the administration of one element may be priorto, concurrent to, or subsequent to the administration of the otheragent(s).

Examples of other active ingredients that may be administered incombination with compositions of the present invention, and eitheradministered separately or in the same agricultural composition,include, but are not limited to:

(1) antifungal agents, such as azoxystrobin and myclobutanil;

(2) plant growth regulators, such as dikegulac-sodium andtrinexapac-ethyl;

(3) herbicides, such as prodiamine and glyphosate;

(4) insecticides, such as bifenthrin and malathion; and

(5) acaricides, such as avermectin and kelthane.

The agricultural compositions of the present invention may contain about0.1 to 95 percent by weight Ilicicolin H.

It is another aspect of the present invention to provide methods oftreating, controlling, or preventing fungal infections on anagricultural product comprising applying to the agricultural product orplant the present agricultural compositions of Ilicicolin H. Thecompositions of the present invention may be applied using a variety ofmethodologies, including soil drench, seed treatment, granular andfoliar spray. With regard to the treatment of seeds, the compositionsmay alternatively be applied directly to the seed before planting of theseed or applied to the locus or soil in which the seeds are sown priorto the planting of the seed.

In such methods of the present invention the concentration of IlicicolinH to be applied to the agricultural product is about 0.001 to 1 percentby weight. In a class of this method the concentration of Ilicicolin Hto be applied to the agricultural product is about 0.001 to 0.01 percentby weight.

Another aspect of the present invention relates to the total amount ofIlicicolin H to be applied per area of the field. In one class of thisaspect about 2 to about 1000 grams of Ilicicolin H are to be applied peracre. In a subclass of this class about 5 to 500 grams of Ilicicolin Hare to be applied per acre. In a second class of this aspect about 0.025kilograms to about 5 kilograms of Ilicicolin H are to be applied perhectare. In a subclass of this second class about 0.05 kilograms toabout 1 kilogram of Ilicicolin H are to be applied per hectare.

Another aspect of the present invention relates to the use of IlicicolinH and the compositions of the present invention to treat, control, orprevent fungal infections on an agricultural product.

It is another aspect of the present invention to provide for the use ofIlicicolin H in the manufacture of a composition for use in treating,controlling, or preventing a fungal infection on an agriculturalproduct.

The present invention also relates to a food, feed, or agriculturalproduct treated with a composition of the present invention.

The present invention also relates to a process for the treatment of anagricultural product which comprises applying a composition of thepresent invention to such agricultural product or plant.

The present invention also relates to the use of a fermentation brothderived from strains of Cylindrocladium ilicicola and Gliocladium roseumas a live cell or cell suspension to treat, control, or prevent a fungalinfection on an agricultural product. Such fermentation broths containthe antifungal agent Ilicicolin H which need not be isolated, purified,and re-formulated for use as an agricultural fungicide.

These and other aspects will become readily apparent from the detaileddescription of the invention which follows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the use of the naturallyoccurring antifungal Ilicicolin H to control the growth of agriculturalpests including, but not limited to, Botryotinia fuckeliana, Glomerellalagenarium, Mycosphaerella arachidis, Zymoseptoria tritici,Gaeumannomyces graminis, Monographella nivalis, Thanatephorus cucumeris,Sclerotinia sclerotiorum, and Puccinia. Ilicicolin H can be used tocontrol a variety of plant diseases and can be applied in a variety ofmethodologies, including soil drench, seed treatment, granular andfoliar spray. It can be readily formulated into a variety ofcompositions, including, but not limited to, emulsifiable concentrates,inverse emulsions, microemulsions, dispersible granules, wettablepowders, dusts, and granules.

Ilicicolin H offers several advantages over existing syntheticfungicides. Since it is derived from natural sources, it already occursin the environment and biological processes to degrade the compound arealready present in the environment. Unlike many conventional fungicides,Ilicicolin H does not contain any heavy metals or halogens and thereforeis totally biologically degradable. Moreover, Ilicicolin H has adifferent mechanism of action from existing commercial fungicides andtherefore can be used to reduce fungicide resistance found in certainplant diseases.

The term “agricultural product” as used herein is to be understood in avery broad sense and includes, but is not limited to, cereals, e.g.,wheat, barley, rye, oats, rice, sorghum, and the like; beets, e.g. sugarbeet and fodder beet; pome and stone fruit and berries, e.g. apples,pears, plums, apricots, peaches, almonds, cherries, strawberries,raspberries and blackberries; leguminous plants, e.g. beans, lentils,peas, and soybeans; oleaginous plants, e.g. rape, mustard, poppy, olive,sunflower, coconut, castor-oil plant, cocoa, and ground-nuts;cucurbitaceae, e.g. pumpkins, gherkins, melons, cucumbers, squashes, andaubergines; fibrous plants, e.g. cotton, flax, hemp, and jute; citrusfruit, e.g. oranges, lemons, grapefruits, mandarins, and limes; tropicalfruit, e.g. papayas, passion fruit, mangos, carambolas, pineapples,bananas, and kiwis; vegetables, e.g. spinach, lettuce, asparagus;brassicaceae, such as cabbage and turnips, carrots, onions, tomatoes,potatoes, seed potatoes, hot and sweet peppers; laurel-like plants, e.g.avocado, cinnamon, camphor tree; or products, such as maize, tobacco,nuts, coffee, sugarcane, tea, grapevines, hops, rubber plants, as wellas ornamental plants, e.g. cut flowers, roses, tulips, lilies,Narcissus, crocuses, hyacinths, dahlias, Gerbia, carnations, fuchsias,chrysanthemums, and flower bulbs, shrubs, deciduous trees and evergreentrees such as conifers, plants and trees in greenhouses. It includes,but is not limited to, plants and their parts, fruits, seeds, cuttings,cultivars, grafts, bulbs, tubers, root-tubers, rootstocks, cut flowersand vegetables.

The following Examples are provided to illustrate the invention and arenot intended to be construed as limiting the scope of the invention inany manner.

Example 1

Ilicicolin H was evaluated in mycelial growth tests in artificial mediaagainst Pythium dissimile, Alternaria solani, Botryotinia fuckeliana,and Gibberella zeae at rates of 20 parts per million (ppm). Mycelialgrowth or disease inhibition was assessed visually and scored using a 3band system (0, 55, and 99 where 99=total inhibition of hyphalgrowth/disease development, 55=partial inhibition, 0=no inhibition), 4to 14 days after inoculation depending on the assay. Scores are givenfor numbered replicates, and average scores for the replicates (avg).Results are presented in Table 1 below. Ilicicolin H was active againstBotryotinia fuckeliana but not active against Pythium dissimile,Alternaria solani, and Gibberella zeae.

TABLE 1 Disease control efficacy Fungal species (average of two repeatexperiments) Pythium dissimile 0 Alternaria solani 0 Botryotiniafuckeliana 99 Gibberella zeae 0

Example 2

Ilicicolin H was also evaluated against several pathogens on leaf-pieceassays at the rate of 100 ppm for Uromyces viciae-fabae on bean andZymoseptoria tritici on wheat, and at the rate of 200 ppm forPhytophthora infestans on tomato. The compound was applied prior toinoculation with the pathogens.

Test species Host Rate (ppm) Zymoseptoria tritici Wheat 100 Phytophthorainfestans Tomato 200 Uromyces viciae-fabae Bean 100

Mycelial growth or disease inhibition was assessed visually and scoredusing a 3 band system (0, 55 and 99 where 99=total inhibition of hyphalgrowth/disease development, 55=partial inhibition, 0=no inhibition), 4to 4 days after inoculation depending on the assay. Scores are given fornumbered replicates, and average scores for the replicates (avg).Results are presented in Table 2. Ilicicolin H was active againstPhytophthora infestans and Zymoseptoria trici and active in one repagainst Uromyces viciae-fabae.

TABLE 2 Disease control efficacy Fungal species (average of two repeatexperiments) Phytophthora infestans (tomato) 99 Zymoseptoria tritici(wheat) 99 Uromyces viciae-fabae (bean) 49

Example 3

In order to test the effectiveness of Ilicicolin H on a variety of plantpathogens at different concentrations, leaf disks or leaf segments ofvarious plant species were cut from plants grown in the greenhouse. Thecut leaf disks or segments were placed in multi-well plates (24-wellformat) onto water agar. The leaf disks were sprayed with a testsolution before (preventative) or after (curative) inoculation with theappropriate pathogen. Iliciolin H was prepared as a DMSO solution (max.10 mg/ml) which was diluted to the appropriate test concentration with0.025% Tween 20 immediately before spraying. The inoculated leaf disksor segments were incubated under defined conditions (temperature,relative humidity, light, etc.) according to the respective test system.A single evaluation of disease level was carried out 3 to 9 days afterinoculation (depending on the pathosystem) and percent disease controlrelative to the untreated check leaf disks or segments was thenassessed. The results are shown in Tables 3 and 4 below. Ilicicolin Hdemonstrated activity against Puccinia recondia.

TABLE 3 Treatment Pathogen Host timing Phytophthora infestans TomatoPreventative Plasmopara viticola Grapevine Preventative Blumeriagraminis f. sp. tritici Wheat Preventative (Erysiphe graminis) Pucciniarecondita Wheat Preventative Puccinia recondita Wheat CurativeMagnaporthe grisea (Pyricularia oryzae) Rice Preventative Phaeosphaerianodorum (Septoria nodorum) Wheat Preventative Pyrenophora teres BarleyPreventative Alternaria solani Tomato Preventative

TABLE 4 Leaf-piece Leaf-piece Leaf-piece test rates test rates testrates (200 ppm), (60 ppm), (20 ppm), Fungal species efficacy efficacyefficacy Phytophthora infestans (tomato- 0 0 0 preventative) Plasmoparaviticola (grapevine- 0 0 0 preventative) Blumeria graminis f. sp.tritici 0 0 0 (Erysiphe graminis) (wheat- preventative) Pucciniarecondita (wheat- 90 50 0 preventative) Puccinia recondita (wheat- 0 0 0curative) Phaeosphaeria nodorum 0 0 0 (Septoria nodorum) (wheat-preventative) Pyrenophora teres (barley- 0 0 0 preventative) Alternariasolani (tomato- 0 0 0 preventative) Magnaporthe grisea (rice- 0 0 0preventative)

Example 4

Suspensions of mycelial fragments or conidia of a fungal species,prepared either freshly from liquid cultures of the fungus or fromcryogenic storage, were directly mixed into nutrient broth. DMSOsolutions of Ilicicolin H (max. 10 mg/ml) were diluted with 0.025% Tween20 by a factor of 50, and 10 μL of this solution was pipetted into amicrotitre plate (96-well format). The nutrient broth containing thefungal spores/mycelia fragments was then added to give a finalconcentration of Ilicicolin H. The test plates were incubated in thedark at 24° C. and 96% relative humidity. The inhibition of fungalgrowth was determined photometrically after 2-7 days, depending on thepathosystem, and percent antifungal activity relative to the untreatedcheck was calculated. The results are shown in Tables 4 and 5 below.Ilicicolin H demonstrated activity against Botryofinia funckeniana,Glomerella lagenanium, Mycosphaerella arachidis, Zymnoseptoria tritici,Gaeumanomyces graminis, Monographella nivalis, Thanalephorus cucumeisand Sclerotinia sclerotiorum.

TABLE 4 Pathogen Pythium ultimum Botryotinia fuckeliana (Botrytiscinerea) Glomerella lagenarium (Colletotrichum lagenarium)Mycosphaerella arachidis (Cercospora arachidicola) Zymoseptoria triticiGaeumannomyces graminis Monographella nivalis (Microdochium nivale)Fusarium culmorum Thanatephorus cucumeris (Rhizoctonia solani)Sclerotinia sclerotiorum

TABLE 5 Liquid- Liquid- Liquid- culture culture culture test rates testrates test rates (20 ppm), (2 ppm), (0.2 ppm), Fungal species efficacyefficacy efficacy Pythium ultimum 0 0 0 Botryotinia fuckeliana 100 70 0(Botrytis cinerea) Glomerella lagenarium 100 50 0 (Colletotrichumlagenarium) Mycosphaerella arachidis 100 100 0 (Cercospora arachidicola)Zymoseptoria tritici 100 100 90 Gaeumannomyces graminis 100 90 0Monographella nivalis 100 100 0 (Microdochium nivale) Fusarium culmorum0 0 0 Thanatephorus cucumeris 70 50 0 (Rhizoctonia solani) Sclerotiniasclerotiorum 50 20 0

Example 5

For the production of Ilicicolin H, Cylindrocladium ilicicola is grownand cultivated as described in Hayakawa et al., “Ilicicolins,antibiotics from Cylindrocladium ilicicola,” Journal of Antibiotics1971, 24, 653-4. Gliocladium roseum is grown and cultivated as describedin Junker et al., “Scale-Up Studies on a Defined Medium Process forPilot Plant Production of Illicicolin by Gliocladium roseum,”Biotechnology Progress 2001, 17, 278-286. The fermentation broth fromproducer strains is lyophilized to remove water to afford a dry powder.The cells in the fermentation broth is broken if necessary by additionof organic solvent such as methanol, ethanol, acetone, and the like, andthen evaporated to dryness to afford a powder. The fermentation brothobtained directly or after breaking the cells with solvent is sprayedonto the plant by foliar treatment, mixed with seeds, or used as a soilinoculant. In addition, broth powders generated using either of the twomethods is mixed with appropriate agent(s) and sprayed by foliartreatment, mixed with seeds, or used as a soil inoculant.

Examples of Formulations of Ilicicolin H for Agricultural Use

Specific embodiments of a formulation of Ilicicolin H for use inagriculture are provided below. The final amounts (wt. %) of concentratecomponents are set forth in Table 6 below.

TABLE 6 Example Example Example Example Example Ingredient Function A BC D E Tetrohydrofurfuryl Solvent 70.91 64.19 69.19 35.59 36.59 alcoholEthyl lactate Solvent 0 0 0 35.60 36.60 Butyl ether derivative Non-ionic0 3.00 0 0 0 of EO/PO block surfactant copolymer (Toximul ® 8320)Tristyrylphenol Non-ionic 11.10 8.50 0 11.00 11.00 ethoxylate withsurfactant approximately 16 moles ethoxylation (Soprophor ® BSU)Polyethylene glycol Non-ionic 2.02 0 0 2.00 0 dilaurate (PEG 400DL)surfactant Tristyrylphenol Anionic 0 8.50 15.00 0 0 ethoxylate phosphatesurfactant ester (Soprophor ® 3D33) Ilicicolin H technical Active 5.925.86 5.86 5.86 5.86 (purity 97%) ingredient

While the invention has been described and illustrated in reference tospecific embodiments thereof, those skilled in the art will appreciatethat various changes, modifications, and substitutions can be madetherein without departing from the spirit and scope of the invention. Itis intended therefore that the invention be limited only by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

1-4. (canceled)
 5. A method for treating or controlling a fungalinfection on an agricultural product comprising the application to saidagricultural product a composition comprising an agriculturallyeffective amount of Ilicicolin H and an agriculturally acceptablecarrier. 6-9. (canceled)
 10. The method according to claim 5 whereinsaid agricultural product is a plant or seed.
 11. The method accordingto claim 5 wherein the application is directly to the agriculturalproduct.
 12. The method according to claim 11 wherein the application isby foliar spray. 13-15. (canceled)